Project Summary Stem cells rely on asymmetric cell division to maintain their stem cell population and give rise to differentiated cells. In order for stem cells to continue to maintain their cell identity post asymmetric division, they rely on their epigenetic information. Epigenetic information can be defined as factors that regulate gene expression changes without altering the primary DNA sequences. The epigenetic regulator, Polycomb group (PcG), is a conserved transcription repressive complex that regulates many stem cell lineages. A decrease in PcG function can lead to a loss of stem cells, while an increase of PcG activity can cause overproliferation of progenitor cells in stem cell lineages and thus tumor growth. As a result, accurate control of PcG activity is necessary for normal development, tissue homeostasis and regeneration. By using the Drosophila male germline as a model system, our lab investigates epigenetic regulation on stem cell maintenance, proliferation, and differentiation. Within the Drosophila testis, there are two populations of stem cells, the germline stem cells (GSCs) and the cyst stem cells (CySCs). Previous studies from the Chen Laboratory reported that PcG genes act in the CySCs lineage to regulate GSC identity and activity, providing a new understanding of non-cell- autonomous regulation by an epigenetic regulator. More recently, the Polycomb (Pc) subcomponent of PcG has been found to localize into a single major punctum in early stage germ cells within the Drosophila germ line. In addition, the Pc punctum is also enriched for DNA replication initiation and histone locus body components. To determine the function of this enriched Pc punctum, first, I would like to characterize the main structural component of the Pc punctum by a series of genetic disruption assays to determine the key Pc punctum components for nucleation and their interactions. Then, I will study how the Pc punctum is able to form by purifying Pc protein to perform biophysical assays such as droplet formation and single molecule FRET to investigate the molecular interactions of different punctum components to create the structure. Finally, I will investigate the function of this punctum in vitro and in vivo, by examining how mutations in Pc affect its ability to phase separate in vitro and then adding this Pc variant to a mutant background in vivo, to examine its phenotypic effects on the germline. The results of these studies should help me to gain insight as to the formation of the Pc punctum and its potential role in DNA replication initiation in germ cells. Through this work I hope to enhance our basic knowledge in the stem cell and chromatin biology fields to be applied in the future to therapeutic treatment of cancers. The ample scientific resources and supportive scientific community provided by Johns Hopkins University, and the support I will receive from my co-mentors, will allow me to complete my thesis work and become a well-rounded scientist ready to pursue a post-doctoral position at a research focused institution.